The chromosomes of all organisms are subject to the processes of transcription, DNA replication/repair and segregation to daughter cells. Chromatin, the complex of chromosomal DNA and proteins in eukaryotes, has a profound impact on these fundamental processes, and defects in chromatin regulation are associated with a variety of human diseases including cancer. The chromatin fiber, which consists of repeating subunits of nucleosomes, is used as the foundation for the assembly of complex higher-order structures involving additional levels of condensation, looping and folding of chromosomes. The goal of this research is to contribute to an understanding of the assembly of higher-order chromatin using a combination of biochemistry and genetics in the single-celled microbe Saccharomyces cerevisiae. The focus is the formation of "silenced chromatin" at a locus called HAIR that serves as a paradigm for the structure, function, and heritable nature of higher-order chromatin. HMR silencing encompasses -4 kb of chromosomal DNA and is controlled by a small DNA element called a silencer that functions by binding directly to silencer-binding proteins such as the Origin Recognition Complex (ORC) that in turn recruit specialized silencing proteins, such as Sirlp, through protein-protein interactions. This silencer-protein-complex "nucleates" the formation of silenced chromatin, which includes the binding of specialized non-histone proteins, such as Sir3p to the chromatin fiber. A specific Sirlp-ORC interaction is central to formation of the silencer-protein-complex. ORC also functions in the more general and essential process of replication initiation at the hundreds of "generic" (non-silencer) replication origins through out the genome. Thus some mechanism must confine a functional Sirlp-ORC interaction to silencers. We postulate that ORC-DNA interactions and additional chromatin-binding proteins insure that a stable physical interaction between Sirlp and ORC occurs only at silencers. In addition, we postulate that ORC-DNA interactions within HMR suppress ORC's function in replication initiation. Lastly, we postulate that the Sir3p binds to nucleosomes at HMR and contributes directly to "remodeling" the HMR chromatin fiber into a larger domain of "higher-order" chromatin.